Triose Phosphate Isomerase

From Proteopedia

Revision as of 15:33, 20 March 2010

Template:STRUCTURE 2ypi

Overview

Triose Phosphate Isomerase (TPI or TIM)is a ubiquitous enzyme with a molecular weight of 56 kD which catalyzes the reversible interconversion of the triose phosphate isomers dihydroxyacetone phosphate (DHAP) and D-glyceraldehyde-3-phosphate , an essential process in the glycolytic pathway. More simply, the enzyme catalyzes the isomerization of a ketose (DHAP) to an aldose GAP also referred to as PGAL. In regards to the two isomers, at equilibrium, roughly 96% of the triose phosphate is in the DHAP isomer form; however, the isomerization reaction proceeds due to the rapid removal of GAP from the subsequent reactions of glycolysis. TPI is an example of a catalytically perfect enzyme, indicating that for almost every enzyme-substrate encounter, a product is formed and that this interaction is only limited by the substrate diffusion rate. Other catalytically perfect enzymes include carbonic anhydrase, acetylcholinesterase, catalase and fumarase. In addition to its relevance in glycolysis, TPI is also involved in metabolic biological processes such as gluconeogenesis, pentose phosphate shunt and fatty acid biosynthesis among others.

Mechanism

TPI catalyzes the transfer of a hydrogen atom from carbon 1 to carbon 2, an intramolecular oxidation-reduction reaction. This isomerization of a ketose to an aldose proceeds through an cis-enediol intermediate. This isomerization proceeds without the need for any cofactors and the enzyme confers a 10⁹ rate enhancement relative to the nonenzymatic reaction involving carboxylate ion.^[1]

Acid Base Catalysis

TPI carries out the isomerization reaction through an acid base mediated mechanism involving . First the PGA molecule is initially attracted to the enzyme active site by the positively charged , with the resulting electrostatic interactions stabilizing the substrate. plays the role of the general base catalyst by abstracting a proton from the pro(R) position of carbon 1. However, the carboxylate groupof Glutamate 165 alone does not possess the basicity to abstract a proton and requires , the general acid, to donate a proton to C-2 to stabilize the negatively charge C-2 carbonyl group, effectively forming the endediol intermediate. At this point in the mechanism, Glutamate 165 acts as a general acid by donating its proton the C-2, while Histidine 95 now acts as a general base by abstracting a proton from the hydroxyl group of C-1. The final step in the reaction is the formation of the GAP isomer product while glutamate and histidine are returned to their original forms, regenerating the enzyme. Additionally, the reaction mechanism of the methylglyoxal forming enzyme methylglyoxal synthase (MGS) is believed to be similar to that of triosephosphate isomerase. Both enzymes utilize DHAP to form an enediol(ate) phosphate intermediate as the first step of their reaction pathways; however, the second catalytic step in the MGS reaction pathway features the elimination of phosphate and collapse of the enediol(ate) to form methylglyoxal rather then reprotonation to form the isomer glyceraldehyde 3-phosphate as seen in TPI.^[2]

Inhibitors of Triose Phosphate Isomerase

Although a highly studied enzyme, there are relatively few effective inhibitors of TPI. From a pharmaceutical perspective, if TPI structures differ greatly between humans and microorganisms such as Plasmodium or Trypanosoma, whose growth rely heavily or entirely on glycolysis, inhibition may be a strong therapeutic target.^[3] Two irreversible inhibitors, halo-acetone phosphate and glycidol phosphate, act by labeling active site residues. There are several weak reversible inhibitors of TPI including 3-Phosphoglycerate, glycerol phosphate and phosphoenol pyruvate, with K_i values ranging from 0.2-1.3 mM.^[4] Additionally, two competitive inhibitors of TPI have shown moderate effectiveness including the 'high-energy intermediate (HEI)analogue' Phosphoglycolohydroxamate (K_i = 6-14 μM) and the 'transition-state analogue phosphoglycolic acid (K_i = 3 μM).^[5]

Structure & Function

Template:STRUCTURE 2ypi Triose Phosphate Isomerase is a member of the all alpha and beta (α/β) class of proteins and it is a homodimer consisting of two nearly identical subunits each consisting of 247 amino acids and differing only at their N-terminal ends. Each TPI monomer contains the full set of catalytic residues; however, the enzyme is only active in the oligomeric form. ^[6] Therefore, dimerization is essential for full function of the enzyme even though it is not believed that any cooperativity exists between the two active sites.^[7] Each subunit contains 8 exterior surrounding 8 interior , which form a conserved structural domain called a closed alpha/beta barrel (αβ) or more specifically a , a domain estimated to be present in 10% of all enzymes. Characteristic of most all TIM barrel domains is the presence of the enzyme's active site in the lower loop regions created by the eight loops that connect the C-terminus of the beta strands with the N-terminus of the alpha helices.TIM barrel proteins also share a structurally conserved phosphate binding motif, with the phosphate either coming from the substrate or from cofactors. ^[8].

Ω Loop 6

As mentioned earlier, TPI is a catalytically perfect enzyme and accomplishes this largely due to its ability to suppress or prevent undesired side reactions such as the decomposition of the enediol intermediate into methyl glyoxal and orthophosphate, a process which is 100 fold faster in solution than the desired isomerization. TPI is able to prevent this undesired reaction by trapping and stabilizing the charged endiol(ate) intermediate in the active site through the use of a flexible 11 residue Ω loop referred to as containing residues 168-178^[9], residue numbers variable with regards to species. Loop 6 can be further divided into a 3-residue N-terminal hinge, a rigid loop tip spanning 5-residues and a 3-residue C-terminal hinge. The complete closure of this loop, a movement of roughly 7 Ǎ for the tip of the loop (C_α of Thr172) and occurring on a microsecond timescale, is facilitated by hydrogen bonding between the hydroxyl group of Tyrosine 208 and the amine nitrogen of Alanine 176 as well as hydrogen bonding between Serine 211 and Glycine 173. As mentioned above, the loop shuts when the enediol is present, effectively shielding both ligand and catalytic residues from solvent exposure, and reopens when the isomerization is complete. Site-directed mutagenesis experiments substituting a Phenylalanine for the Tyrosine resulted in a 2400-fold decrease in catalytic activity. ^[10] Additionally, extensive mechanistic and kinetic experiments involving Trypanosoma brucei, a parasitic protist causing sleeping sickness in humans, has revealed the structural and functional importance of a proline residue at position 168 in conjunction with transmitting the signal of ligand binding to the conformational change of the catalytic glutamate residue (Glu167 in T.brucei) and the subsequent proper loop 6 closure.^[11] Specifically, the proline residue is positioned at the beginning of loop 6 as to aid in the catalytic glutamate side chain flipping from the inactive swung-out to the active swung-in conformation, facilitating the closure of the loop. Structurally, in the unliganded (open) conformation, the Glu-Pro peptide bond is in the energetically favored trans conformation; however, in the liganded (closed) conformation, the pyrrolidine ring of proline adopts a rare strained planar conformation (9 kJ/mol in vacuo), suggesting that the strain could be important for loop opening and product release, upon completion of the reaction cycle.^[12]

Entropic Effects of Ω Loop 6 Hinges

Similar to the loop spanning residues, the Ω loop 6 hinge residues share high sequence homology amongst species. The role of both the N- and C-terminal hinge regions of Ω loop 6 have been extensively studied including the replacement of conserved hinge residues with glycine, which resulted in a 2500-fold drop in k_cat. The insertion of glycine into the hinge region significantly increases the flexibility of the loop due to glycine's conformational freedom, which in turn allows the loop to sample many more conformations. This has thermodynamic ramifications as these glycine-rich hinge mutants prompted a large entropy gain (+ΔS) compared to WT, effectively altering the entropic activation energy. Specifically, WT TPI is able to overcome the initial entropic gain (order to disorder), caused by dispelling water molecules from the active site, by forming a more ordered enzyme-substrate complex. Conversely, the glycine-rich hinge mutants again promote an initial entropy gain due to water loss but are unable to pay the entropic penalty due to their inability to bind substrate tightly. Since catalysis will only occur when the closed conformation has been sampled it reasons that the likelihood of sampling this conformation is greatly reduced with the glycine-rich hinge mutants. As its overall biological role in the enzyme, the loop hinges act to limit the motion of the loop which effectively restricts the number of conformations accessible to the enzyme. In this manner, TPI acts like an entropy trap.

Disease

Template:STRUCTURE 2ypi Triose Phosphate Isomerase Deficiency, initially described in 1965, is an autosomal recessive inherited disorder with characteristics ranging from chronic haemolytic anaemia, increased susceptibility to infections, severe neurological dysfunction, and often times death in early childhood.^[13] TPI has been most closely linked to a point mutation at the residue which results in the mutation. A common marker for TPI deficiency is the increased accumulation of dihydroxyacetone phosphate in erythrocyte extracts as a result in the inability of the mutant enzyme to catalyze the isomerization to D-glyceraldehyde-3-phosphate. Recent evidence has indicated that the point mutation does not prove detrimental to the rate of catalysis of the enzyme, but rather effects the ability of the enzyme to dimerize.^[14]

Role in Alzheimer's Disease: Recent discoveries in Alzheimer Disease research has indicated that amyloid beta-peptide induced nitro-oxidative damage promotes the nitrotyrosination of the glycolytic enzyme triosephosphate isomerase in human neuroblastoma cells.^[15] nitro-triosephosphate isomerase was found to be present in brain slides from double transgenic mice overexpressing human amyloid precursor protein as well as in Alzheimer's disease patients. Specifically, the nitrotyrosination occurs on , which are located in close proximity to the catalytic center, and this modification correlates with a reduced isomerase activity. Additionally, according to work done by Francesc Guix and colleagues, nitro-triosphosphate isomerase contributed to the formation of large beta-sheet aggregates in vitro and in vivo.

Evolutionary Conservation

Template:STRUCTURE 2ypi

Due to its role in the glycolysis, an essential process to many organisms, TPI has been isolated and crystallized from several species giving rise to extensive multiple alignment in silico experiments which subsequently provided of TPI. ^[16] Collectively, these tools have determined that TPI has a roughly 50% sequence conservation from bacteria to humans,^[17]

Links

• 1tim (Triose Phosphate Isomerase from Gallus gallus)
• 2ypi (Complex with TPI and PGA substrate from Saccharomyces cerevisiae)
• 2vom (Glu104Asp mutation contributing to TPI deficiency)
• 1wyi (Triose Phosphate Isomerase from Homo sapians)
• 2j27 (Pro168Ala mutation outlining functional role of active site proline)

References

1. ↑ Davenport RC, Bash PA, Seaton BA, Karplus M, Petsko GA, Ringe D. Structure of the triosephosphate isomerase-phosphoglycolohydroxamate complex: an analogue of the intermediate on the reaction pathway. Biochemistry. 1991 Jun 18;30(24):5821-6. PMID:2043623
2. ↑ Saadat D, Harrison DH. The crystal structure of methylglyoxal synthase from Escherichia coli. Structure. 1999 Mar 15;7(3):309-17. PMID:10368300
3. ↑ Fonvielle M, Mariano S, Therisod M. New inhibitors of rabbit muscle triose-phosphate isomerase. Bioorg Med Chem Lett. 2005 Jun 2;15(11):2906-9. PMID:15911278 doi:10.1016/j.bmcl.2005.03.061
4. ↑ Fonvielle M, Mariano S, Therisod M. New inhibitors of rabbit muscle triose-phosphate isomerase. Bioorg Med Chem Lett. 2005 Jun 2;15(11):2906-9. PMID:15911278 doi:10.1016/j.bmcl.2005.03.061
5. ↑ Fonvielle M, Mariano S, Therisod M. New inhibitors of rabbit muscle triose-phosphate isomerase. Bioorg Med Chem Lett. 2005 Jun 2;15(11):2906-9. PMID:15911278 doi:10.1016/j.bmcl.2005.03.061
6. ↑ Rodriguez-Almazan C, Arreola R, Rodriguez-Larrea D, Aguirre-Lopez B, de Gomez-Puyou MT, Perez-Montfort R, Costas M, Gomez-Puyou A, Torres-Larios A. Structural basis of human triosephosphate isomerase deficiency: mutation E104D is related to alterations of a conserved water network at the dimer interface. J Biol Chem. 2008 Aug 22;283(34):23254-63. Epub 2008 Jun 18. PMID:18562316 doi:10.1074/jbc.M802145200
7. ↑ Schnackerz KD, Gracy RW. Probing the catalytic sites of triosephosphate isomerase by 31P-NMR with reversibly and irreversibly binding substrate analogues. Eur J Biochem. 1991 Jul 1;199(1):231-8. PMID:2065677
8. ↑ http://www.ncbi.nlm.nih.gov/Structure/cdd/cddsrv
9. ↑ Joseph D, Petsko GA, Karplus M. Anatomy of a conformational change: hinged "lid" motion of the triosephosphate isomerase loop. Science. 1990 Sep 21;249(4975):1425-8. PMID:2402636
10. ↑ Derreumaux P, Schlick T. The loop opening/closing motion of the enzyme triosephosphate isomerase. Biophys J. 1998 Jan;74(1):72-81. PMID:9449311 doi:10.1016/S0006-3495(98)77768-9
11. ↑ Casteleijn MG, Alahuhta M, Groebel K, El-Sayed I, Augustyns K, Lambeir AM, Neubauer P, Wierenga RK. Functional role of the conserved active site proline of triosephosphate isomerase. Biochemistry. 2006 Dec 26;45(51):15483-94. Epub 2006 Dec 19. PMID:17176070 doi:10.1021/bi061683j
12. ↑ Kursula I, Wierenga RK. Crystal structure of triosephosphate isomerase complexed with 2-phosphoglycolate at 0.83-A resolution. J Biol Chem. 2003 Mar 14;278(11):9544-51. Epub 2003 Jan 9. PMID:12522213 doi:http://dx.doi.org/10.1074/jbc.M211389200
13. ↑ Schneider AS. Triosephosphate isomerase deficiency: historical perspectives and molecular aspects. Baillieres Best Pract Res Clin Haematol. 2000 Mar;13(1):119-40. PMID:10916682
14. ↑ Ralser M, Heeren G, Breitenbach M, Lehrach H, Krobitsch S. Triose phosphate isomerase deficiency is caused by altered dimerization--not catalytic inactivity--of the mutant enzymes. PLoS ONE. 2006 Dec 20;1:e30. PMID:17183658 doi:10.1371/journal.pone.0000030
15. ↑ Guix FX, Ill-Raga G, Bravo R, Nakaya T, de Fabritiis G, Coma M, Miscione GP, Villa-Freixa J, Suzuki T, Fernandez-Busquets X, Valverde MA, de Strooper B, Munoz FJ. Amyloid-dependent triosephosphate isomerase nitrotyrosination induces glycation and tau fibrillation. Brain. 2009 May;132(Pt 5):1335-45. Epub 2009 Feb 27. PMID:19251756 doi:10.1093/brain/awp023
16. ↑ Parthasarathy S, Ravindra G, Balaram H, Balaram P, Murthy MR. Structure of the Plasmodium falciparum triosephosphate isomerase-phosphoglycolate complex in two crystal forms: characterization of catalytic loop open and closed conformations in the ligand-bound state. Biochemistry. 2002 Nov 5;41(44):13178-88. PMID:12403619
17. ↑ Joseph-McCarthy D, Lolis E, Komives EA, Petsko GA. Crystal structure of the K12M/G15A triosephosphate isomerase double mutant and electrostatic analysis of the active site. Biochemistry. 1994 Mar 15;33(10):2815-23. PMID:8130194